INTRODUCTION:

One of the first anticancer medications to be created was a folate inhibitor. Methotrexate (MTX), a similar medication, is today a crucial part of modern ALL therapy. Aminopterin was initially used to induce remission in children with acute lymphoblastic leukemia (ALL) in 1948.

Since 1951, MTX has been used to treat psoriasis and rheumatoid arthritis (RA). However, it was not until the 1980s that MTX therapy for RA became popular. Subsequent research revealed that in patients with severe and chronic RA, it was substantially more beneficial than a placebo. Today, it is also utilized to treat ectopic pregnancy and a number of malignant conditions. The Food and Drug Administration (FDA) authorized MTX as a therapy for RA in 1988. MTX may increase efficacy when taken with other biological disease-modifying antirheumatic medications (DMARDs). In many nations, MTX is a first-line antirheumatic medication since its benefits outweigh its drawbacks.¹

Structure of methotrexate:

Fig.1.1 Structure of methotrexate

IUPAC Name:-(2S)-2-[[4-[(2,4-diaminopteridin-6-yl) methyl-methylamino] benzoyl] amino] pentane dioic acid

Formula: - C₂₀H₂₂N₈O₅

Molar mass: - 454.447 g·mol−1²

HISTORY AND DISCOVERY:

The history of methotrexate (MTX) starts a few years earlier, in 1945, when American researcher of Indian descent Subbarow (forgotten or too little known) isolated folic acid from the liver for the first time in the laboratory of the pharmaceutical company that would later become Lederle. Most people associate this with researchers with names like Farber or Guber.

At this point, it should be stated that Gubner’s 1951 study on the use of methotrexate in rheumatoid arthritis was received at that time with the query “Why employ such a lethal anti-cancer Drug to treat a benign ailment such as rheumatoid arthritis?”

A group of dermatologists from Scandinavia reported a number of serious side effects of methotrexate use in 1973, including liver cirrhosis in psoriasis patients taking the medication multiple times a week. However, they failed to mention that the patients had unfettered access to alcohol and had previously been exposed to arsenic, which was still being used to treat psoriasis at the time.³

PHYSIOCHEMICAL PROPERTIES:

1. Solubility:

Solubility in water: At 25°C, methotrexate dissolves in water just slightly (~0.01mg/mL).

Its carboxylic acid groups deprotonate, making it more soluble in alkaline solutions.

Solubility in Organic Solvents: Almost insoluble in acetone, ether, and chloroform, among other organic solvents.

pH Dependence: Because the carboxyl groups ionize when the pH rises, solubility dramatically increases.⁴

2. Melting point:

Melting Point: Approximately Decomposes at about 195 to 205°C.

When heated, methotrexate breaks down rather than melting smoothly.⁵

3. UV-Visible Absorption:

UV Spectra Characteristics: Methotrexate shows characteristic UV absorption maxima at ~258nm and ~302nm in aqueous solution, depending on pH.⁶

4. Infrared (IR) Spectroscopy:

Key IR Absorption Peaks (KBr disc or Nujol mull):

~3300–3400 cm⁻¹: N-H and O-H stretching

~1700–1720 cm⁻¹: C=O stretching (carboxylic acid group)

~1600 cm⁻¹: Aromatic C=C stretching

~1500–1550 cm⁻¹: N-H bending (primary amines)

~1200–1300 cm⁻¹: C-N stretching (aromatic amines)

These correspond to the various functional groups present: amine groups, carboxylic acid, aromatic rings, and the pteridine ring system.⁷

PHARMACOKINETICS PROPERTIES:

Table.1.1 pharmacokinetics properties of methotrexate

Pharmacokinetics properties
BCS classification	Due to its hydrophobic nature and low permeability, MTX is categorized as a class IV molecule in the Biopharmaceutical Classification System (BCS).⁸
Cmax	MTX maximum plasma concentrations (Cmax) range between 0.3 and 1.6 µmol/L, and occur at a Tmax of 0.75 to 2 h after administration.⁹
Tmax	Methotrexate has a Tmax of 1 to 2 hours. oral doses of 10-15µg reach serum levels of 0.01-0.1µM. The volume of distribution of methotrexate at steady state is approximately 1L/kg.
T half	The half life of low dose methotrexate is 3 to 10 hours in people. High doses of methotrexate have a half-life of 8 to 15 hours. The terminal half-life of methotrexate in pediatric patients with acute lymphoblastic anemia ranges from 0.7 to 5.8 hours. The half-life of methotrexate in pediatric patients with juvenile idiopathic arthritis ranges from 0.9 to 2.3 hours.
Absorption	The bioavailability of methotrexate ranges from 64 to 90%; however, it drops at oral dosages greater than 25 mg because the carrier-mediated transport of the drug becomes saturated.
Distribution	The volume of distribution of methotrexate at steady state is approximately 1L/kg.
Protein binding	Methotrexate is 46.5-54% bound to plasma proteins
Metabolism	In the liver and other organs, folylpolyglutamate synthase converts methotrexate to methotrexate polyglutamate. The glutamyl chains of methotrexate polyglutamates are hydrolyzed by gamma-glutamyl hydrolase, which then transforms them back into methotrexate. Additionally, a tiny quantity of methotrexate is changed into 7-hydroxymethotrexate.
Elimination	About 3% of methotrexate is eliminated as the 7-hydroxylated metabolite, while over 80% of the medication is eliminated unaltered. 8.7–26% of an intravenous dose of methotrexate appears in the bile, with the majority of the drug being eliminated in the urine.¹⁰

MECHANISM OF ACTION:

The drug is absorbed into the cell by human reduced folate carriers (SLC19A1) and forms methotrexate-polyglutamate. Both unaltered methotrexate and methotrexate-polyglutamate inhibit the enzyme dihydrofolate reductase, which catalyzes the conversion of dihydrofolate into tetrahydrofolate, the active form of folic acid. Methotrexate has multiple mechanisms of action, which makes it an effective treatment for a variety of conditions.

The synthesis of DNA and RNA nucleotides requires tetrahydrofolate. Additionally, methotrexate-polyglutamate inhibits thymidylate synthase, which stops DNA synthesis, and purine synthase, which suppresses the de novo synthesis of purine nucleotides. The main reason methotrexate is prescribed as a cancer treatment is because of the cytotoxic effect that these distinct effects combine to create.

Similarly, people with autoimmune illnesses may be prescribed methotrexate due to completely distinct mechanisms of action.

Fig.1.2 Pharmacokinetics properties of methotrexate

SYNTHESIS OF METHOTREXATE:

Methotrexate synthesis involved, a multi-step procedure begins with 2,4,5,6-tetraaminopyrimidine, which is then followed by reactions with 1,1-dichloroacetone and p-(N-methyl)-amino benzoyl-L-glutamic acid.

Synthesis process:

1. Initial Condensation:

The synthesis begins with the condensation of 2,4,5,6-tetraaminopyrimidine with 1,1-dichloroacetone. This reaction, often carried out in the presence of sodium bisulfite at a controlled pH (3.5 to 5) and temperature (10° to 100°C), yields 2,4-diamino-6-methylpteridine.

2. Bromination:

To create 2,4-diamino-6-bromomethylpteridine, the resultant 2,4-diamino-6-methylpteridine is subsequently brominated.

In this stage, a certain amount of bromide is utilized per gram of the pteridine compound, and the bromide is reacted with in a particular reaction medium.

3. Glutamate Coupling as the Last Step:

Lastly, methotrexate is created when 2,4-diamino-6-bromomethylpteridine combines with p-(N-methyl)-amino benzoyl-L-glutamic acid or one of its salts in a polar reaction media.

The amide bond between the pteridine ring and the L-glutamic acid component of the molecule is formed in this phase. 8 science direct.¹²

Fig. 1.4 synthesis of methotrexate

MEDICINAL USES OF DRUG:

Methotrexate (MTX) is a folate antagonist with diverse medical applications, particularly in oncology, autoimmune diseases, and inflammatory disorders.

1. Cancer Chemotherapy:

Mechanism: Methotrexate reduces DNA synthesis, repair, and replication by inhibiting dihydrofolate reductase (DHFR), particularly in cells that divide quickly.

Route – II From: Pyrimidine – 2,4,5,6-tetraamine

Fig.1.5 Synthesis of methotrexate

Indication:

Treatments for acute lymphoblastic leukemia (ALL) include induction and maintenance.

Osteosarcoma: - High-dose MTX combined with leucovorin rescue is a common treatment for osteosarcoma.

Non- Hodgkin lymphoma (NHL) :- R-CHOP and other combination treatments include non-Hodgkin lymphoma (NHL).

Multidrug regimens are used to treat bladder cancer, head and neck cancer, and breast cancer.¹³

2. Rheumatoid Arthritis (RA):

Mechanism: Methotrexate suppresses the immune system and reduces inflammation at low doses, in part via increasing the release of adenosine.

Indications: - RA treatment using a first-line disease-modifying antirheumatic medication (DMARD). used either alone or in combination with other DMARDs such as hydroxychloroquine or sulfasalazine.¹⁴

3. Psoriasis and Psoriatic Arthritis: Reduces epidermal cell proliferation and T-cell activation.

Indications:

Moderate to severe psoriasis (especially plaque-type).

Psoriatic arthritis when skin and joint involvement are significant.¹⁵

4. Ectopic Pregnancy: Inhibits trophoblastic cell division and growth.

Indications:

Unruptured ectopic pregnancy with stable hemodynamics and specific size/hCG criteria.

Medical management alternative to surgery.¹⁶

5. Crohn’s Disease and Inflammatory Bowel Disease (IBD): Immune suppression in inflammatory pathways.

Indications: Second-line immunosuppressant after thiopurines or when biologics are contraindicated.¹⁷

Fig.1.6 FDA Approved uses

ADVERSE EFFECTS:

Gastrointestinal Effects: Nausea, vomiting, and diarrhea are common, especially at higher doses.

Mucositis (inflammation of the mucous membranes, often leading to painful ulcers) is also reported.¹⁸

Hematological toxicity: Leukopenia, anemia, and thrombocytopenia are among the known and potentially dangerous side effects of bone marrow suppression. To control these effects, routine blood monitoring is essential.¹⁹

Liver Toxicity: Methotrexate can cause hepatotoxicity, including increased liver enzymes and more severe diseases including cirrhosis with long-term use. Regular monitoring of liver function is necessary.²⁰

Pulmonary Toxicity: Pneumonitis is one type of interstitial lung disease (ILD) that can cause symptoms including coughing and dyspnea. Although uncommon, this toxicity is severe and may necessitate stopping the medication.²¹

Renal Toxicity: Methotrexate is renally excreted, and impaired renal function can lead to drug accumulation and increase the risk of toxicity. Acute kidney injury (AKI) is a possible complication, especially in those with pre-existing kidney disease.²²

Reproductive Toxicity: Methotrexate is teratogenic and should be avoided during pregnancy. It can cause miscarriage and birth defects.²³

TREATMENT OF OVERDOSE:

Consequences of overdose:

1. Bone marrow suppression

2. Renal toxicity

3. Pulmonary toxicity

4. Gastrointestinal toxicity

Treatment of overdosing:

1. Leucovorin Rescue: Leucovorin (folinic acid) is used to mitigate the toxic effects of methotrexate on normal cells by bypassing the inhibition of folate metabolism caused by methotrexate. The dosage of leucovorin depends on the serum methotrexate level and the time since overdose. It should be administered as soon as possible after the overdose and is often continued for several days, with the exact regimen tailored to the patient's methotrexate levels and clinical response.²⁴

2. Alkalinization of Urine: Methotrexate is excreted via the kidneys, and urinary alkalinization (usually with sodium bicarbonate) can help enhance the renal excretion of methotrexate by increasing its solubility in urine. This is crucial in preventing renal damage. However, excessive alkalinization should be avoided to prevent complications like metabolic alkalosis.²⁵

3. Hemodialysis: Hemodialysis is the most effective method for removing methotrexate from the body, especially when the overdose is severe or the patient has compromised renal function. Hemodialysis can rapidly reduce serum methotrexate levels and prevent further organ damage. Dialysis is particularly indicated when serum methotrexate levels remain high despite other treatments, or if kidney function is severely impaired.²⁶

4. Supportive Care: Hydration: Ensuring adequate fluid intake is essential to support renal function and methotrexate excretion. IV fluids should be administered to maintain hydration and urine output.²⁷

CONTRAINDICATIONS:

Methotrexate is contraindicated for cases with acuity responses to this drug. Pregnant or breastfeeding cases should avoid using methotrexate due to the elevated threat of teratogenicity and excretion into bone milk. In cases of rheumatoid arthritis or psoriasis, it's contraindicated to use methotrexate in cases with habitual liver complaint, cirrhosis, alcoholic hepatitis, or active alcohol use complaint. Methotrexate isn't recommended for cases with HIV/ AIDS, blood dyscrasias, renal dysfunction, or those witnessing radiotherapy.¹¹

Liver disease - chronic liver disease (e.g, cirrhosis hepatitis B/C)

Bone marrow suppression - Existing Anemia, leukopenia, or, thrombocytopenia

Renal impairment - moderate to severe dysfunction

Lung Disease - Interstitial lung Disease, pulmonary fibrosis

INTERACTION:

Since methotrexate is strongly attached to plasma proteins, any medication that removes it from proteins can raise blood levels. It may interact with protein-bound medications such tetracyclines, phenytoin, salicylates, sulfonamides, sulfonylureas, and oral anticoagulants.

Methotrexate levels may rise as a result of any medication that affects renal clearance. Aminoglycosides, neomycin, and probenecid decrease the absorption of MTX, while nonsteroidal anti-inflammatory medications (NSAIDs), salicylates, TMP, penicillin, warfarin, valproate, proton pump inhibitors, cyclosporin, and cisplatin raise the risk of MTX toxicity in the blood.

Because of the prevalence of concurrent prescriptions, NSAIDs and PPIs are the most common interactions.

When methotrexate is taken alongside other drugs, its plasma concentrations can change, raising the possibility of serious side effects and perhaps decreasing the production of active metabolites, which could reduce the drug's therapeutic efficacy. This includes NSAIDs, hepatotoxic or nephrotoxic chemicals, oral antibiotics like neomycin and penicillin, and antifolate medications like dapsone and pyrimethamine. Nitrous oxide should be used with caution in patients on methotrexate because it may intensify the drug's effects on folate metabolism, raising the possibility of serious side effects. Alternative treatments must to be taken into account.

Due to competition for active transport across cell membranes, coadministration of folic acid or its derivatives may lessen the efficacy of methotrexate in the treatment of neoplastic disorders. For best therapeutic results, patients should take folic or folinic acid as directed by their healthcare physician.

Methotrexate may negatively impact humoral and cellular immunological responses to COVID-19 mRNA vaccinations in individuals with immune-mediated inflammatory disorders. Although precise immunogenicity thresholds associated with vaccine effectiveness are still unknown, these results imply that different approaches and more study may be required.¹¹

Table.1.2 Drug – Drug interaction²⁸

Drug	Interaction
Acetylsalicylic acid, Amoxicillin	Reduced excretion of methotrexate (increased toxicity)
Corticosteroid	Increased risk of hematological toxicity
Pyrimethamine	Antifolate effect of methotrexate increased
Sulfamethoxazole + trimethoprim	Antifolate effect of methotrexate increased (avoid concomitant use); risk of methotrexate toxicity increased
Vaccine, live	Avoid use of live vaccines with methotrexate (impairment of immune response)
Nitrous oxide	Increased antifolate effect (avoid concomitant use)

CONVENTIONAL AND NOVEL MARKETED FORMULATION:

Table.1.3. Formulations of methotrexate^29,30

Type	Brand name	Company name	Dose	Prize
Tablet	Folitrax 2.5mg	Ipca laboratories	2.5 mg	₹50 for a pack of 10 tablet
	Mytrexate	Prevego Healthcare	5 mg	₹77.44 for pack of 10 tablet
	Mexate 7.5 mg	Zydus cadila	7.5 mg	₹133.7 for pack of 10 tablet
	Oncotrex 5mg	Sun pharmaceutical industries Ltd	5mg	₹97.50 for pack of 10 tablet
	Imutrex 2.5mg	Cipla Ltd	2.5 mg	₹42.3 for pack of 10 tablet
	Biotrexate 2.5 mg	Biotech pharmaceuticals	2.5 mg	₹49 to 103 for pack of 10 tablet
Injection	Folitrax 20 mg	Ipca laboratories Ltd	20 mg per / ml vial	₹121.75 per vial
	Methocel 50 mg	Celon lab	50 mg	₹47.3 per vial
	Metex 1000mg	Intas pharmaceutical Ltd	1000 mg per vial	1093 per vial

PATENT:

Table.1.4. Patent on drug methotrexate^{31,32,33,34,35,36}

Sr.no	Patent no.	Name of inventor	Title	Description	Year of grant	Year of expiry
1.	US9,259,427 B2	Paul Robert Benians (Rosemont Pharmaceuticals Ltd.)	Methotrexate compositions for oral administration	Oral methotrexate liquid formulation with improved patient compliance.	February 16, 2016	February 16, 2036
2.	US10,231,927 B2	Paul Robert Benians	Methotrexate compositions for oral administration	Improved liquid methotrexate composition for pediatric and elderly use	March 19, 2019	March 19, 2039
3.	US11,969,503 B2	Paul Robert Benians	Methotrexate formulations and methods of treatment	Newly optimized oral methotrexate formulations for better dosing control.	April 30, 2024	April 30, 2044
4.	US11,129,833 B2	Christopher Henshall (Thera kind Ltd.)	Methotrexate pharmaceutical composition	Methotrexate compositions intended for pediatric use	September 8, 2021	September 8, 2041
5.	US11,129,899 B2	Ornin Ruangwattanasuk (S.S. Manufacturing Co., Ltd.)	Methotrexate derivatives and methods of treatment	Novel methotrexate derivatives for improved therapeutic effect	September 28, 2021	September 28,2041
6.	US7,229,623 B2	Ronald S. Horwitz	Parenteral methotrexate compositions	Stable injectable methotrexate compositions.	June 12, 2007	June 12, 2027

CONCLUSION:

One well-known folate antagonist that has a big influence on the treatment of autoimmune diseases and cancer is methotrexate (MTX). It was first used to treat leukemia in the 1940s, but it has since developed into a mainstay treatment for diseases like psoriasis, rheumatoid arthritis, ectopic pregnancy, and several types of cancer. Notwithstanding its efficacy, methotrexate's limited therapeutic index and a number of severe side effects, including as hepatotoxicity, pulmonary problems, and hematologic toxicity, restrict its use. It also requires cautious administration due to many medication interactions and contraindications. However, it is a first-line treatment in many regimens due to its cost-effectiveness and variety of modes of action. Formulation innovations, such as oral liquid formulations, have increased patient compliance, particularly in the elderly and pediatric populations. Methotrexate is still an essential medication with careful supervision and ongoing study with continued research and careful monitoring, methotrexate remains a vital agent in modern pharmacotherapy.

REFERENCE:

1. Hamed K M, et al. Overview of Methotrexate Toxicity: A Comprehensive Literature Review. Cureus. 14(9): e29518. DOI 10.7759/cureus.29518

2. Methotrexate - Wikipedia https://en.m.wikipedia.org/wiki/Methotrexate

3. Anand, N. Malaviya, Landmark papers on the discovery of methotrexate for the treatment of rheumatoid arthritis and other systemic inflammatory rheumatic diseases: a fascinating story, International Journal of Rheumatic Diseases. 2016; 19: 844–851; https://onlinelibrary.wiley.com/doi/abs/10.1111/1756-185X.12862

4. Methotrexate sodium Injection USP monograph – United States Pharmacopeia

5. https://pubchem.ncbi.nlm.nih.gov/compound/Methotrexate#section=InChI

6. Silverstein, Webster, Kiemle - Spectrometric Identification of Organic Compounds

7. Indian Pharmacopoeia 2010 - volume 2

8. Giri, B.R.; et. al Improved Bioavailability and High Photostability of Methotrexate by Spray-Dried Surface-Attached Solid Dispersion with an Aqueous Medium. Pharmaceutics. 2021; 13(1): 111; https://doi.org/10.3390/pharmaceutics13010111

9. Bedoui Y, et.al Methotrexate an Old Drug with New Tricks. Int J Mol Sci. 2019 Oct 10; 20(20): 5023. doi: 10.3390/ijms20205023

10. Methotrexate: Uses, Interactions, Mechanism of Action | DrugBank Online https://go.drugbank.com/drugs/DB00563

11. https://www.ncbi.nlm.nih.gov/books/NBK556114/

12. https://www.sciencedirect.com/topics/chemistry/methotrexate

13. Chabner, B. A., and Longo, D. L. (2011). Cancer Chemotherapy and Biotherapy: Principles and Practice. Lippincott Williams and Wilkins.

14. Singh, J. A., et al. American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis. Arthritis and Rheumatology. 2016; 68(1): 1–26.

15. Menter, A., et al. Guidelines of care for the management of psoriasis and psoriatic arthritis. J Am Acad Dermatol. 2009; 61(3): 451–485.

16. Barnhart, K. Ectopic Pregnancy. New England Journal of Medicine. 2009; 361(4): 379–387.

17. Lichtenstein, G. R., et al. ACG Clinical Guideline: Management of Crohn's Disease in Adults. Am J Gastroenterol. 2018; 113(4): 481–517.

18. McCune, J. S., and Clark, M. A. The Pharmacology of Methotrexate in Rheumatology and Cancer. Clinical Pharmacology and Therapeutics. 2011; 90(1): 1-5.

19. Lichtenstein, G. R., and Yajnik, V. Methotrexate-induced Hematologic Toxicity in Inflammatory Bowel Disease. Journal of Clinical Gastroenterology. 2003; 37(4): 243-249.

20. Miele, L., and Cicala, M. Hepatotoxicity of Methotrexate in Rheumatologic Therapy. Digestive and Liver Disease. 2006; 38(2): 116-123.

21. Kähler, C. M., and Eberhard, L. Pulmonary Toxicity Induced by Methotrexate in Patients with Rheumatoid Arthritis: A Systematic Review. Journal of Rheumatology. 2012; 39(5): 912-917.

22. Weiner, M., and Press, O. W. Renal Toxicity of Methotrexate: Mechanisms and Management. Seminars in Oncology. 2009; 36(4): 289-297

23. Moxley, G. M., and Smith, P. Methotrexate in the Treatment of Rheumatoid Arthritis and Pregnancy Considerations. American Journal of Obstetrics and Gynecology. 2007; 197(5): 457.e1-457.e7.

24. Fisher, B. Folinic acid rescue for methotrexate overdose. Seminars in Oncology. 1994; 21(6): 689-694.

25. Chan, S., et al. Management of methotrexate overdose. Journal of Clinical Pharmacology. 2017; 57(9): 1050-1057.

26. Hayes, T. G., et al. Management of high-dose methotrexate toxicity. Journal of Oncology Pharmacy Practice. 2019; 25(2): 408-418.

27. McAlister, D. R., et al. Methotrexate overdose: A review of current management practices. Pharmacotherapy. 2018; 38(12): 1262-1274.

28. National Formulary of India 4th Edition 2011, 648-695

29. https://www.netmeds.com/prescriptions/methotrexate -1ml

30. https://www.apollopharmacy.in/medicine/methotrexate-2-5mg-tablet?srsltid=AfmBOoolW11FeNqm6stqOtZrPvcpBktzlvEGqrc8hYjUhXLyi9bxO-zq

31. Benians, P. R. (2016). Methotrexate compositions for oral administration. U.S. Patent No. 9,259,427. Washington, DC: U.S. Patent and Trademark Office. https://patents.google.com/patent/US9259427B2

32. Benians, P. R. (2019). Methotrexate compositions for oral administration. U.S. Patent No. 10,231,927. Washington, DC: USPTO. https://patents.google.com/patent/US10231927B2

33. Benians, P. R. (2020). Methotrexate compositions for oral administration. U.S. Patent No. 10,610,485. Washington, DC: USPTO. https://patents.google.com/patent/US10610485B2

34. Henshall, C. (2021). Methotrexate pharmaceutical compositions. U.S. Patent No. 11,129,833. Washington, DC: USPTO. https://patents.google.com/patent/US11129833B2

35. Ruangwattanasuk, O. (2021). Methotrexate derivatives and methods of treatment. U.S. Patent No. 11,129,899. Washington, DC: USPTO. https://patents.google.com/patent/US11129899B2

36. Horwitz, R. S. (2007). Parenteral methotrexate compositions. U.S. Patent No. 7,229,623. Washington, DC: USPTO. https://patents.google.com/patent/US7229623B2

Received on 09.05.2025 Revised on 20.06.2025

Accepted on 26.07.2025 Published on 06.10.2025

Available online from October 13, 2025

Asian J. Pharm. Res. 2025; 15(4):418-424.

DOI: 10.52711/2231-5691.2025.00065

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.